1. Technical Field
The present invention relates to mutants of the tegument protein of human cytomegalovirus (CMV) known as CMVpp65. The mutants desirably do not exhibit the protein kinase activity which is associated with the native pp65 protein but retain its desirable immunologic target characteristics.
2. Description of the Background Art
The CMV genome is relatively large (about 235k base pairs) and has the capacity to encode more than two hundred proteins. CMV is composed of a nuclear complex of double-stranded DNA surrounded by capsid proteins having structural or enzymatic functions, and an external glycopeptide- and glycolipid-containing membrane envelope. CMV is a member of the herpes virus family and has been associated with a number of clinical syndromes.
Human cytomegalovirus is not only a significant cause of morbidity in persons undergoing immunosuppressive therapy, but remains the major infectious cause of congenital malformations and mental retardation (26, 38, 53) (see the appended list of References for the identification of references cited throughout this specification). Although improved antiviral chemotherapy is becoming available for management of CMV infection, the large number of congenital infections (approximately 35,000 newborns per years in the U.S. (9)) underscores the need for an effective CMV vaccine (37), especially one which can be used safely in healthy persons. Attenuated and recombinant live virus vaccine approaches have been proposed, but safe use of these types of vaccines in healthy populations remains to be shown. Subunit vaccines are attractive because they have the potential to boost the immune system against certain viral proteins without risking viral infection or viral recombination.
CMV infection is widespread and persistent, and can become reactivated and clinically evident in the immunosuppressed patient. Because human cytomegalovirus is relatively common, yet is associated with extremely serious health conditions, a considerable effort has been made to study the biology of the virus with the aims of improving diagnosis of the disease as well as developing preventative and therapeutic strategies.
It would be highly desirable to deliver an effective vaccine derived from CMV that would impart immunity to persons at risk of CMV disease such as a bone marrow transplant (BMT) recipient, a solid organ recipient, a heart patient, an AIDS patient or a woman of child-bearing years. No such vaccine presently is commercially available, however.
Cell-mediated immunity (CMI) plays an essential role in recovery from acute CMV infection and in the control of persistent CMV infection. The generation of cytotoxic T lymphocytes (CTL) is a most important factor in limiting CMV disease. Several proteins encoded by CMV are known to be recognized by the cellular immune system and elicit CTL. Borysiewicz et al. (2) first described the role of specific CMV proteins in CTL induction. The non-virion, immediate early proteins of CMV (CMV-IE), as well as the envelope glycoprotein, CMVgB, activate CTL function, however, the internal matrix proteins of the virus, CMVpp65 and CMVpp150, are more prevalent immune targets. CMVpp65 is the immunodominant protein: 70-90% of all CMV-specific CTL recognize this protein.
CMVpp65 is not essential for virus replication, therefore it may function to facilitate host cell changes important to virus spread. CMVpp65 has emerged as the primary target of CMV-specific CTL. Because it is a structural virus protein, it is available as an immune target immediately after infection, in the absence of virus replication. Thus, CMVpp65 is a preferred target for the cellular immune system.
CMVpp65 is known to interact with the cellular polo-like kinase-1 that is present at high levels during cellular mitosis (15). It contains redundant nuclear localization signals (17, 43) and becomes associated with nuclear lamina and condensed chromosomes during infection (8, 42). Thus, CMVpp65 clearly has nuclear and chromosomal trafficking ability that could represent an unknown risk if the protein were expressed in normal cells.
CMVpp65 also has been reported to have endogenous serine/threonine phosphotransferase activity (2, 3, 31, 32, 35, 41), however, it lacks several of the recognizable protein kinase (PK) consensus domains (21) (see FIG. 1). The kinase activity of CMVpp65 remains incompletely understood, but certain consensus sequences conserved threonine/serine/tyrosine PK catalytic domain are found within the 173 amino acid carboxy-terminal region of CMVpp65 (45). Only three subdomains align properly with these conserved residues of the catalytic domain (21) but two other subdomains are present. These CMVpp65 motifs consist of the catalytic subdomain I (amino acids 422-427; EXEXXE; SEQ. ID NO: 1), subdomain II (amino acid 436; K), and subdomain VIB (amino acids 543-545; RDL. See FIG. 1. The sequences not in precise alignment are subdomain VIII (amino acids 463-465; APE, upstream from subdomain VIB and subdomain XI (carboxyl terminal amino acid 460; R).
PK activity plays an important role in the regulation of normal and transformed cell growth (7, 11, 25) and is important in viral regulation of cellular functions (29) and in regulation of virus transcription, DNA synthesis, and virion assembly (18, 36, 47, 51, 52). Because Protein kinases play an important role in the regulation of both normal and malignant cell growth (46), DNA vaccines (13, 34) that depend on the expression of intact CMVpp65 also could pose problems associated with introduction of PK activity into normal cells. The growth effects of increased kinase activity in healthy cells could limit the use of intact CMVpp65 as a vaccine, especially in children and women of child-bearing age. Therefore, new CMVpp65-derived sequences which could be used in DNA vaccines, yet which lack the undesired activities of the native protein would be highly useful.